Avoid the deleterious effects of mispriming at lower temperatures with the QuantiNova Probe PCR Kit. Based on a novel, antibody-mediated, hot-start mechanism, the kit enhances the specificity and efficiency of probe-based real-time PCR to provide accurate, singleplex or duplex, cDNA or gDNA analysis on various real-time PCR cyclers. The added convenience of extreme stability for up to 100 hours at room temperature, without a need for any cooling agent, makes it ideal for handling of high-throughput samples and automated workflows. An in-built tracking system for visual identification of correct pipetting gives you absolute peace of mind while the extreme sensitivity for even low target amounts ensures reliable qPCR results every time.

The QuantiNova Probe PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Sensitive and robust detection of a single target copy.

[A] The QuantiNova Probe PCR Kit was used to detect the single-copy gene, CFTR, in leukocyte genomic DNA using 30, 3, 0.3, and 0.03 ng gDNA, corresponding to 104 to 10 copies, and the results were plotted to create a calibration curve. The plot of copy number versus CT value demonstrates high linearity. A master mix sufficient for 60 reactions was set up and 180 pg template gDNA, corresponding theoretically to 1 target copy per reaction was added to the master mix. The master mix was pipetted into 60 wells. However, due to statistical variations, some wells had more than 1 target copy while others had none. The calibration curve was used to determine the actual number of copies within each of the 60 wells. [B] The expected number of copies per well was calculated theoretically using Poisson's equation and compared to the actual number of copies determined using the calibration curve. There was a high concordance between the single-copy number results obtained using the calibration curve and Poisson's equation, demonstrating the high sensitivity and robustness of the QuantiNova Probe PCR Kit.

Consistent results on various real-time PCR cyclers

Expression of EGFR in HeLa cells was analyzed. RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit. Reactions were run in triplicate using 10-fold dilutions of the cDNA (10 ng to 0.01 ng) on various cyclers fully exploiting the fast-cycling capabilities of each cycler. The QuantiNova Probe PCR Kit provides consistent sensitivity, reproducibility, and efficiency on the tested cyclers, despite the varying cycling conditions.

Superior results with challenging assays.

The performance of the QuantiNova Probe PCR Kit was compared to a probe PCR kit from supplier B on a Bio-Rad CFX Connect cycler [A] and a probe PCR kit from supplier L [B] on a ViiA7 cycler. Reactions were run in triplicate using 10-fold dilutions of plasmid DNA (106–102 copies per reaction) and a TaqMan assay detecting a 500 bp amplicon and using a minor-groove binding probe. The QuantiNova Probe PCR Kit provides significantly lower CT values, higher reproducibility, and higher reaction efficiency, compared to the probe PCR kits from suppliers B and L.

Comparable results in duplex and singleplex PCR.

Duplex and singleplex PCR was performed on a Bio-Rad CFX96 cycler using TaqMan Assays for GAPD and TNF with the QuantiNova Probe PCR Kit. Ten-fold dilutions of leukocyte DNA (from 100 ng to 10 pg) were used as templates and reactions were run in triplicate. [A] Overlay of the TNF amplification curve for the singleplex and duplex reactions and [B] overlay of the GAPD amplification curve for the singleplex and duplex reactions. The plots demonstrate the comparability and reliability of the results for singleplex and duplex amplification using the QuantiNova Probe PCR Kit.

Principle of the novel QuantiNova hot-start mechanism.

QuantiNova DNA Polymerase is kept in an inactive state by QuantiNova Antibody and QuantiNova Guard until the initial heat activation step.

Built-in visual control for correct pipetting

The master mix supplied with the QuantiNova Probe PCR Kit contains an inert blue dye that does not interfere with the real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When the template nucleic acid, diluted with the QuantiNova Yellow Template Dilution Buffer, is added to the master mix, the color of the solution changes from blue to green (see figure Built-in pipetting control), providing a visual indication of correct pipetting and reaction setup.

Reaction stability of up to 100 hours

The real-time PCR mix can be stored at 30°C for up to 100 hours without impairing the performance of subsequent reactions (Table 1). The outstanding stability, even after extended storage at room temperature without the use of any cooling agent, makes the QuantiNova Probe PCR Kit ideal for high-throughput reaction setup and plate-stack handling (see table "Effect of storage on reaction stability").

Table 1. Effect of storage on reaction stability

Mean CT values

QuantiNova Probe PCR Kit

Probe PCR kit from supplier L

Amount in ng

0 h

100 h

0 h

100 h

10

24.37

24.49

24.35

27.49

1

27.78

27.90

27.77

30.97

0.1

31.16

30.98

31.37

34.48

No template control

Not detected

Not detected

Not detected

Not detected

Table 1. Comparison of CT values before and after 100 hours storage at 30°C. Real-time PCRs including master mix, template cDNA, primers, and probes for TNF were incubated at 30°C for 100 hours and run in the real-time PCR together with freshly prepared reactions. Triplicate reactions of 3 different template amounts were tested. In contrast to the probe PCR kit from supplier L, the CT values provided by the QuantiNova Probe PCR Kit did not vary after extended storage of the samples at 30°C.

Accurate and robust detection of a single DNA molecule

The high sensitivity of the QuantiNova Probe PCR Kit results in accurate and robust detection of even a single target copy (see figure Sensitive and robust detection of a single target copy). The special master mix supplied with these kits enables accurate quantification of 2 targets having widely differing abundance in a single tube. This saves time, money, and reduces the amount of sample material needed. Moreover, the duplex PCR data obtained is comparable with that obtained from a singleplex PCR (see figure Comparable results in duplex and singleplex PCR).

Superior performance for difficult targets

The unique combination of QIAGEN's proprietary and well-proven buffer technology along with the new QuantiNova DNA Polymerase, QuantiNova Antibody, and QuantiNova Guard, ensures real-time PCR success at the first attempt, without the need for costly and time-consuming optimization, even with challenging real-time PCR assays (see figure Superior results with challenging assays).

Highly consistent results on all real-time cyclers

The QuantiNova Probe PCR Kit can be used on any real-time cycler. ROX is provided in a separate tube and can be added if using a cycler that uses ROX as a passive reference dye. The kit delivers highly consistent results among different cyclers in terms of sensitivity, reproducibility, and efficiency. The consistency of the results is maintained, despite varying fast-cycling capacities of the different cyclers that result in different cycling protocols (see figure Consistent results with various real-time PCR cyclers).

Principle

The QuantiNova Probe PCR Kit delivers singleplex or duplex, cDNA or gDNA analysis with highest specificity because of a novel, antibody-mediated, hot-start mechanism (see figure Novel antibody mediated hot-start mechanism). At low temperatures, the QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and a novel additive, QuantiNova Guard, that stabilizes the complex. This improves the stringency of the hot-start and prevents extension of nonspecifically annealed primers and primer–dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated enabling the PCR amplification.

Procedure

QuantiNova Probe PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template gDNA or cDNA, primers, and probe to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. ROX passive reference dye is provided as a separate tube and the concentrations of the dye can be adjusted depending on the type of cycler used, enabling use of the QuantiNova Probe PCR kits on virtually any real-time cycler. Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Applications

QuantiNova Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets and quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent.